The major objective of this proposal is to determine the mechanism by which alpha-adrenergic agents cause a release of calcium from the mitochondria and an increase of the cytosolic free Ca2 ion concentration in isolated hepatocytes. We will examine the hypothesis that turnover of phosphatidylinositol in the plasma membrane is a primary event following hormone-receptor binding, and that this process is concerned with generating an alpha-adrenergic second messenger which causes alterations of the intracellular calcium homeostasis. A specific goal of the project will be to determine the chemical identity and mechanism of action of the postulated alpha-adrenergic second messenger. The possibility will also be assessed that phosphatidylinositol metabolism is a secondary or parallel event more directly connected with the production of arachidonate and subsequent synthesis of prostaglandins. Relative activities of diacylglycerol kinase and lipase will be measured, together with the effects of hormonal stimulation on diacylglycerol, arachidonic acid and prostaglandin production. A further specific goal will be to determine whether other hormones such as glucagon and beta-adrenergic agents, which exert their effects primarily through cAMP-dependent mechanisms, alter cytosolic/mitochondrial free Ca2 ion concentrations or intracellular calcium distribution. Likewise, possible insulin effects on intracellular calcium homeostasis will be assessed. A third specific goal will be to correlate hormonally or A-23187 induced changes of cytosolic free Ca2 ion with the phosphorylation of intracellular proteins and increased Ca2 ion binding to calmodulin. A fourth specific goal will be to investigate the possible role of changes in cytosolic and/or mitochondrial free Ca2 ion, induced by acute glucagon and adrenergic stimulation, diabetes and high protein diets, in the regulation of glucose and urea synthesis. For these studies, emphasis will be placed on the use of branched chain alpha-keto-acids as substrates, with and without addition of ammonia and ornithine. Initially, the studies will be performed mainly with isolated rat hepatocytes, and the more promising aspects will be extended to adipocytes and kidney tubules.